CHANGES IN CONFORMATION OF PHOSPHORYLATED PROTEINS

已知蛋白质磷酸化控制着广泛的细胞事件，如：代谢、细胞分裂、膜转运和基因表达等。然而，磷酸化调控的生物物理基础只在两个系统中被研究过。结合光谱-分子建模方法将提供必要的结构信息，以帮助理解磷酸化在蛋白质功能中的抑制或激活作用。抑制或激活蛋白质功能的一种可能机制是，磷酸化可能诱导构象的改变，从而影响其与其他蛋白质或生物分子的相互作用。本研究中使用的模型蛋白是中心蛋白。中心蛋白是一种普遍存在的蛋白质，与中心粒/基体、中心体和有丝分裂纺锤杆有关，因此是微管组织中心（MTOC）的一个组成部分，负责将染色体分配给子细胞。最近的研究结果表明，在细胞分裂过程中中心体复制过程中，中心体蛋白磷酸化形式的激活作用是必不可少的。我们的目标是：(1)在细菌系统中过度表达Human 1,2和Chlamydomonas centrin，从而获得生物物理研究所需的丰富蛋白质来源。(2)采用多种色谱技术对中心蛋白进行分离和表征，并通过SDS-PAGE、western blot、氨基酸序列和飞行时间（TOF）质谱鉴定其纯度。(3)利用蛋白激酶A （PKA）和三磷酸腺苷（ATP）体外磷酸化中心蛋白，以获得足够量的生物物理表征。(4)利用傅里叶变换红外光谱（FT-IR）和圆二色性（CD）研究磷酸化和未磷酸化的中心蛋白构象。(5)利用二维红外相关性对蛋白磷酸化和非磷酸化状态的光谱进行比较。(6)通过分子模拟技术模拟这些结果。这种跨学科的研究磷酸化中心蛋白的方法将使我们能够在磷酸化调节的蛋白质中建立构象和功能之间的关系。这项研究的意义将是提供分子水平的信息，这将有助于理解所研究的细胞事件，如细胞分裂和不受控制的细胞分裂，如细胞癌。长期目标是利用振动光谱研究位点特异性诱变产生的变异蛋白，并通过核磁共振确定这些蛋白的结构。

Protein phosphorylation is known to control a wide range of cellular events such as: metabolism, cell division, membrane transport and gene expression among others. However, the Biophysical basis of regulation by phosphorylation has only been studied in two systems. A combined spectroscopic-molecular modeling approach will provide the necessary structural information to aid in the understanding of the inhibitory or activating effect of phosphorylation in protein function. One possible mechanism for the inhibitory or activating effects on protein function is that phosphorylation may induce a change in conformation and thus effect its interaction with other proteins or biological molecules. A model protein used in this study is centrin. Centrin has been found to be a ubiquitous protein associated with centrioles/basal bodies, centrosomes, and mitotic spindle poles and therefore is an integral component of the microtubule organizing center (MTOC) which is responsible for distributing the chromosomes to the daughter cells. Recent findings suggests an activating effect on centrin in its phosphorylated forms which is essential in centrosomal duplication during cell division. Our aims are: (1) To over-express Human 1, 2 and Chlamydomonas centrin in a bacterial system allowing for an abundant source of protein required for the biophysical study. (2) To isolate and characterize centrin using various chromatographic techniques and conform its purity via SDS-PAGE, western blot analysis, amino acid sequence and time of flight (TOF) mass spectrometry. (3) To phosphorylate centrin in vitro using protein kinase A (PKA) and adenosine triphosphate (ATP) in sufficient quantities for Biophysical characterization. (4) To investigate phosphorylated and un-phosphorylated centrin conformation using Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD). (5) To use two-dimensional infrared correlation for the comparison of spectra obtained for proteins in their phosphorylation and un-phosphorylated states. (6) To model these results via molecular modeling techniques. This inter-disciplinary approach to the study of phosphorylated centrin will enable us to establish a relationship between conformation and function in a protein regulated by phosphorylation. The implications of such a study will be to contribute molecular level information which will aid in the understanding of the cellular events studied such as cell division and uncontrolled cell division such as in cell carcinomas. Long-term goals are to study variant proteins generated by site-specific mutagenesis using vibrational spectroscopy and to determine the structure of these proteins via NMR.